Abstract

Background

The spadetail (spt) gene of zebrafish is expressed in presomitic mesoderm and in neural cells previously
suggested to be Rohon-Beard neurons. The mechanism(s) generating the apparently irregular
rostrocaudal distribution of spt-expressing cells in the developing CNS is unknown.

Results

spt-expressing neural cells co-express huC, a marker of neurons. These cells also co-express the genes islet-1, -2 and -3 but not valentino. The islet-1 gene expression, irregular distribution and dorsolateral position of spt-expressing cells in the developing CNS are characteristic of dorsal longitudinal
ascending (DoLA) interneurons. Shortly after their birth, these neurons extend processes
rostrally into which spt mRNA is transported. At 24 hours post fertilisation(hpf), spt-expressing neurons occur most frequently at rostral levels caudal of the 5th-formed somite pair. There is no apparent bias in the number of spt-expressing cells on the left or right sides of embryos. Extended staining for spt-transcription reveals expression in the dorsocaudal cells of somites at the same
dorsoventral level as the spt-expressing neurons. There is frequent juxtaposition of spt-expression in newly formed somites and in neurons. This suggests that both types
of spt-expressing cell respond to a common positional cue or that neurons expressing spt are patterned irregularly by flanking somitic mesoderm.

Conclusions

spt-expressing cells in the developing CNS appear to be DoLA interneurons. The irregular
distribution of these cells along the rostrocaudal axis of the spinal cord may be
due to "inefficient" patterning of neural spt expression by a signal(s) from flanking, regularly distributed somites also expressing
spt.

Background

The spinal cord of vertebrates shows no apparent morphological metamerism. However,
the pattern of motor and sensory axonal projection from the spinal cord shows a metameric
distribution that is patterned by the flanking somites [1,2].

In developing zebrafish, both metameric and non-metameric patterns of neuron distribution
can be observed. When primary motoneurons first arise in the developing ventral spinal
cord, three such cells are present per hemisegment [3,4]. Mutation of the gene spadetail (spt) causes changes in somite formation that affect this pattern of motoneuron formation.
This shows that motoneuron patterning is controlled by signals from the somites [5-7]. In contrast, the Rohon-Beard sensory neurons in the dorsal central nervous system
(CNS) show no segmental distribution and are not affected by mutations affecting somite
formation [5]. However, mutations such as bmp2b/swirl, bmp7/snailhouse affecting signalling by members of the bone morphogenic protein (BMP) family, [8]) and changes in Notch signalling [9][10][11] can affect the number/differentiation of these cells.

Rohon-Beard neurons, when they arise, are sufficiently numerous to be found adjacent
to every somite (i.e. in each "hemisegment"). However, a third type of neural cell
distribution exists with less than one cell per hemisegment. For example, dorsal longitudinal
ascending (DoLA) interneurons are found at a frequency of 0.06 per hemisegment for
the 5th- to 8th-formed flanking somite pairs in embryos at 18 hpf [12]. The mechanisms that control these irregular distributions are unknown.

The spt mutation was originally described by Kimmel et al. in 1989 [13] as a γ ray-induced mutation affecting trunk development including somite formation.
Closer analysis of the effect of this mutation on development has shown that spt controls convergence movements and the differentiation fate of mesodermal precursors
of the trunk [13-17].

The locus for spt mutations was identified by Griffin et al. in 1998 [18]. They showed that the spt gene encodes a T-box protein similar to those encoded by the Xenopus gene Xombi (also known as Antipodean, BraT or VegT) and the chick gene Tbx6L. spt is transcribed in caudal paraxial mesoderm before its differentiation to somitic
mesoderm. spt is also expressed in irregularly distributed neural cells that have been suggested,
on the basis of their position and distribution, to be Rohon-Beard neurons [19].

In the work described in this paper, we show that the neural cells expressing spt have the characteristics of DoLA interneurons. We then examine the distribution of
spt-expressing neurons on the rostrocaudal axis and on the left and right sides of embryos.
Intriguingly, we have discovered low-level expression of spt in the dorsocaudal extremities of newly formed somites that corresponds in dorsoventral
level and, frequently, rostrocaudal position, to newly formed neurons expressing spt. This distribution of spt expression suggests the possible existence of an "inefficient" mechanism producing
an irregular pattern of neuron distribution based on a regularly patterned flanking
structure (somitic mesoderm).

Results

Neural spt-expressing cells have the characteristics of DoLA neurons

Cells expressing spt in the developing central nervous system have previously been suggested to be Rohon-Beard
neurons [18,19]. To confirm their neuronal nature, we double-stained embryos for spt expression and the neuronal marker gene huC[20]. We observed coexpression of spt with huC confirming that these cells are neurons (Figure 1D).

Figure 1. Whole mount in situ transcript hybridisation analysis of the expression of spt and other genes in the tail and trunk of zebrafish embryos at approximately 22 hpf.
In all images, dorsal is up and rostral is to the left. An apparently irregular rostrocaudal
distribution of spt-expressing cells is seen in the developing CNS rostral to the domain of expression
in the presomitic mesoderm of the extending tail (A). Boxed areas in A indicate parts
of the image magnified in B and C. Shortly after their birth, these cells extend a
process rostrally (indicated by a black asterisk in B) into which spt transcript is transported. spt is expressed in newly formed somites in a restricted region, the "somitic trail"
(bracketed in C), at the same dorsoventral level as spt-expressing cells in the developing CNS (black arrowheads in any panel). The spt-expressing cells in the developing CNS (red stain) co-express huC, a marker of neurons (blue stain in D). A probe that identifies cells transcribing
val (blue stain) shows that the spt-expressing neurons (red stain) are not identical with these (E). Transcription of
the isl-1 gene (see F) is seen dorsally in Rohon-Beard neurons (black arrows in any panel),
and ventrally in motoneurons (white arrow). Intermediate between these two levels
are DoLA neurons that also express isl-1 (black arrowhead). Double staining with isl-1 (blue) and spt (red) shows that these intermediate-level neurons express spt (G). Costaining of spt (red) with isl-2 (blue in H) and isl-3 (blue in I) shows that the DoLA neurons also apparently express these genes, although
the onset of expression occurs more rostrally than for isl-1. Scale bars equal 100 μm in A, B, C and F and 20 μm in D, E, G, H, I.

To test the idea that spt-expressing neurons are Rohon-Beard neurons we double-stained embryos for expression
of spt and the islet (isl)-1, -2 or -3 genes [6,7,21] or valentino (val, [22]) that have been stated to be expressed in these cells. Interestingly, the spt-expressing neurons also express all three known isl genes but not val (Figures 1E,1G,1H,1I). In embryos at 22 hours post fertilisation (hpf, at 28.5°C), spt-expressing cells express isl-1 from the moment of their first detection at the caudal end of the developing CNS.
isl-2 and isl-3 coexpression with spt is more easily visible at more rostral levels. In every case, the cells co-expressing
the spt and isl genes are located just ventral to dorsally located cells expressing isl genes alone, i.e. Rohon-Beard neurons. The isl-1 expression, rostrocaudal distribution and dorsolateral position of these cells are
characteristic of DoLA interneurons [6,21]. We cannot state with certainty that all DoLA neurons express spt, only that all DoLA neurons expressing isl-1 also appear to express this gene. Contrary to an earlier report [7], we observed expression of isl-2 and isl-3 in these interneurons. This might be explained by difficulty in distinguishing DoLA
neurons from Rohon-Beard neurons at the rostral levels where isl-2 and -3 expression is more easily observed.

spt mRNA is transported into neurite-like structures

Soon after their differentiation in the central nervous system, a rostrally-projecting
process of the spt-expressing neurons can be observed to contain spt mRNA (Figure 1B). This process may, in fact, become the future ascending axon of the DoLA neurons.
The transport of spt mRNA into this process presumably is an active rather than passive process since
other mRNAs, such as those of huC and the islet genes, are not similarly localised (data not shown).

We attempted to observe the pattern of axonal projection from spt-expressing neurons at later times after their differentiation. We stained embryos
at 22 hpf to reveal both spt-transcription and the presence of acetylated tubulin (that labels axons). Confocal
imaging of spt-expressing neurons in the region of the spinal cord dorsal to the yolk extension
showed that these cells lie alongside the dorsal longitudinal fasciculus (DLF, Figure
2). Their proximity to the DLF obscured the pattern of axonal projection from these
cells. We did not observe the presence of spt transcript in axons near these cells.

Figure 2. Close association of neurons expressing spt with the dorsal longitudinal fasciculus. Images shown are projections of serial 0.5
μm optical sections through a 22 hpf embryo stained to reveal spt transcripts (red) and acetylated tubulin (green) that marks axons. The cell shown
lies in that part of the developing spinal cord midway along the yolk extension. Rostral
is to the left in both images. A shows a lateral projection with dorsal to the top.
B shows a dorsal projection with medial to the bottom and lateral to the top. The
size bar in A indicates 10 μm. B has an identical rostrocaudal dimension but the mediolateral
dimension is compressed. The size bar in B indicates 10 μm in the mediolateral dimension.

Dorsoventral and rostrocaudal correspondence of caudal spt expression in the somitic mesoderm and developing CNS

Extended staining for spt expression allowed us to observe spt mRNA in recently-formed somites at 24 hpf just rostral to the previously observed,
high-level expression of spt in the presomitic mesoderm. This expression is not present throughout the somites
but, rather, only at the same dorsoventral level as spt-expressing cells in the developing spinal cord. From a lateral perspective, this
gives the impression of a "trail" of spt-expressing cells in the somitic mesoderm left behind by the extending tail tip (Figures
1C, 3A,3B,3D).

Figure 3. The juxtaposition of spt expression in newly formed somites and the developing CNS at approximately 22 hpf.
In all images dorsal is uppermost and rostral is to the left. A, B and C are views
from one embryo. A and B show the appearance from a lateral view of the tail in the
region of the "somitic trail" of spt expression. A black asterisk indicates the most recently formed somite. spt expression is concentrated to the dorsocaudal extremity of somites. In an optical
(DIC) section through the same embryo viewed from a dorsolateral perspective (C),
the basal lamina separating the developing CNS and the somitic mesoderm can be seen
clearly (arrowheads). Cells expressing spt in the developing CNS (black arrows) are juxtaposed to somitic cells expressing spt (white arrows). The "somitic trail" region of a second embryo is shown in D (lateral
view) and E (dorsolateral view). The black asterisk in E indicates a neural cell expressing
a lower level of spt. Scale bars equal 20 μm.

The somitic expression of spt is strongest in the dorsocaudal cells of these structures (Figure 3). Observation of this region from a dorsolateral perspective shows that cells expressing
spt in the developing spinal cord most commonly form so that they are in direct juxtaposition
with these cells across the basal lamina (Figures 3C,3E; at least 76% of observed cases, n = 25). However, they do not form adjacent to every
somite. This distribution suggests that: 1) the spt-expressing neurons are either generated in response to signals from the dorsocaudal
cells of each somite or, 2) that neural and somitic cells express spt in response to a common patterning signal(s). In either case, an "inefficient" stimulation
of neural cells to transcribe spt would result in the observed distribution of spt-expressing neurons.

Occasionally, neural cells transcribing lower levels of spt can be observed adjacent to the most posterior somites (see asterisk in Figure 3E). We have not observed such cells at more rostral levels so these might represent
cells in the process of activating spt transcription. Alternatively, neural cells transcribing spt at lower levels might be lost or might repress spt transcription later in spinal cord development.

The earliest formation of spt-expressing neurons

The somitic expression of spt at 24 hpf is only seen in the most recently formed somites. We wished to observe
whether newly born spt-expressing neurons are always flanked by spt expression in somitic mesoderm, and to determine the earliest time at which spt-expressing neurons could be observed.

To gain an indication of the time at which spt-expressing neurons might first arise, we observed the somitic juxtaposition of the
most rostral spt-expressing neuron in 11 embryos at approximately 24 hpf. The majority of the embryos
(n = 10) possessed at least 6 somite pairs rostral to the most rostral spt-expressing neural cell (Figures 4A,4B). Only one embryo showed a lower number (at least 5 rostral somite pairs). Thus,
spt-expression in the spinal cord is flanked by the region of somitic mesoderm that shows
slower somite formation (occurring after the initial rapid formation of the first
six somite pairs, [23]). Since the 5th somite pair forms at approximately 12 hpf (at 28.5°C), we examined embryos between
12 hpf and 16 hpf for spt staining in the CNS. The CNS primordium is relatively flattened at this time and
the basal laminae separating CNS, mesoderm and individual somites are difficult to
observe in fixed embryos. Nevertheless, the earliest time at which we could observe
spt expression confidently in the developing CNS was 15.5 hpf (13 somite pairs). At 16
hpf (14 somite pairs), five of six embryos examined for which spt-expressing neurons could be seen had at least 9 somite pairs rostral to the most
rostral spt-expressing neuron (see Figure 5). This observation implies that the spt-expressing neurons at more rostral positions (i.e. adjacent to the 6th to 9th somite pairs) differentiate at later times or that spt-expressing cells migrate rostralwards after their birth (see later). At 16 hpf, the
spt-expressing neural cells are also flanked by low level spt expression in somites (white arrowheads in Figure 5). Thus, low level somitic expression of spt occurs during most of somitogenesis. Low level spt expression is observable at 14.5 hpf in laterocaudal cells. However, we could not
determine whether these cells were neural or mesodermal (data not shown).

Figure 4. Lateral views of two embryos (A and B) at approximately 24 hpf stained to reveal spt transcription. Dorsal is up and rostral is to the left. DIC microscopy was used to
reveal somite boundaries. Consequently, spt-expressing cells in the developing CNS are not seen clearly because they lie in a
different focal plane. However, the most rostral cell in each embryo is indicated
by a white arrrowhead. The most rostral visible discernible somite is indicated by
a white arrow. In both cases there are 6 somites rostral to the most rostral spt-expressing neuron. Scale bars equal 100 μm.

Figure 5. Early spt expression in the developing CNS and somites. A and C show lateral views of two embryos
at 16 hpf. Rostral is up and dorsal is to the right. B shows a dorsal view of the
embryo in A. Rostral is up. White arrowheads indicate the most rostral somitic domain
of spt transcription visible. Black arrowheads indicate the most rostral neural cell expressing
spt. For B, the light source was concentrated behind the yolk to give greater visibility
of staining. All images are composites of smaller images. Scale bars equal 100 μm.

Analysis of left-right bias in spt-expressing neuron number

The irregular distribution of spt-expressing neurons may conceal a left or right bias in the number of these neurons.
To investigate this we examined the numbers of neurons on the left and right sides
of 48 embryos at 24 hpf. The mean number of cells on the left sides of embryos was
found to be 10.5 with a standard deviation of 2.1. The mean number of cells on the
right sides of embryos was found to be 10.7 with a standard deviation of 1.9. The
differences in the mean number of spt-expressing neurons on the left and right sides of the embryos is considerably smaller
than the standard deviations of left and right. This argues against any left-right
bias.

The analysis above might not reveal a left or right bias when the variability in the
number of spt-expressing neurons in each embryo is high. Thus, we also examined the difference
in the numbers of spt-expressing neurons between the left and right sides of individual embryos. For each
of the 48 embryos, the number of spt-expressing cells on the left of the embryo was subtracted from the number on the
right. The mean difference was +0.2 with a standard deviation of 2.0. Since the standard
deviation is far larger that the mean difference, this also argues against any left
or right bias in spt-expressing neuron number. Finally, we tested whether there is simply a tendency for
an absolute difference in the numbers of spt-expressing cells to exist between the two sides of the embryo, regardless of any
left-right bias. The mean absolute bilateral difference for the 48 embryos was 1.5
cells. The standard deviation for this value was 1.4. Thus, there is no significant
difference in the numbers of spt-expressing cells between the two sides of embryos.

Preferred positions of spt-expressing neurons on the rostrocaudal axis

While the distribution of spt-expressing neurons along the rostrocaudal axis of the spinal cord appears to be irregular,
preferred positions may, nevertheless, exist. To analyse this, 20 embryos were fixed
at 24 hpf and stained to reveal expression of spt. The left and right sides of the trunk and tail of the embryos were then photographed
under differential interference contrast (DIC) optics to show simultaneously the spt-expressing neurons and the boundaries between the flanking somitic tissue. We then
counted the neurons occurring adjacent to each particular somite on the left and right
sides of the embryo. Since we have shown that there is no left-right bias in the number
of spt-expressing neurons, we combined the data from the two sides. The number of somite
pairs present in embryos at 24 hpf can vary [23], as can the visibility in fixed embryos of the most anterior somite boundaries and
the most recently formed somite boundaries. Therefore, to make the results from each
embryo comparable, we identified the somite pair directly dorsal to the most caudal
extent of the yolk extension as somite level 0. We then numbered the other somite
pairs according to this reference point (Figure 6). Somite pairs rostral to somite level 0 were given a "+" designation while caudal
somite pairs were given a "-" designation. The mean number of cells present at each
somite level was then calculated (Table 1).

Figure 6. Diagram of somite level designations relative to the caudal tip of the yolk extension
in a 24 hpf embryo.

A tendency to higher numbers of cells at rostral somite levels is evident. The highest
mean number observed was at somite level +11 (1.9 cells per embryo for left and right
sides combined). At 24 hpf, somite level +11 commonly corresponds to the 7th somite pair formed. Lower numbers of spt-expressing neurons are observed at somite levels caudal to somite level 0 (commonly
the 18th somite pair formed). However, there is great variability between embryos in the number
of cells at any somite level (as indicated by the large standard deviation values
in Table 1). The increase in cell number at rostral levels is not explained by the increase
in the rostrocaudal dimension of somites as they mature since the segmental pattern
of neuron distribution in the spinal cord expands correspondingly [2]. The higher number of spt-expressing neurons found rostral to somite level 0 could be due to: 1) continuing
birth of these neurons at rostral positions as the CNS develops, 2) programmed cell
death of neurons at caudal positions, or 3) rostralwards migration of neurons after
their birth. Two observations support the last possibility. First, the mean number
of spt-expressing neurons along the entire rostrocaudal axis per embryo was determined for
76 embryos at 24 hpf (21.4 neurons, standard deviation 3.4) and 45 embryos at 30 hpf
(22.7 neurons, standard deviation 2.9). Somitogenesis ends at approximately 24 hpf
but differentiation along the rostrocaudal axis continues in a rostral to caudal manner.
Thus, any later, rostral generation of spt-expressing neurons or programmed cell death of caudal neurons as spinal cord development
continues after 24 hpf might be expected to alter the average number of neurons by
a greater number than that observed. Second, ipsilateral juxtaposition of spt-expressing neurons (which we defined as instances in which the cell bodies of the
neurons appear to contact each other) occurred for 5.5% of cells in the region of
somite levels -11 to +4, but for 11.3% of these cells in the region of somite levels
+5 to +12. These data, together with the observation of greater neuron numbers at
rostral levels, suggest that these neurons accumulate at rostral levels due to rostralwards
migration after their birth.

No spt-expressing cells were observed rostral of somite level +13, commonly corresponding
to the 5th somite pair formed. This could be an artefact of the low number of embryos for which
these somite levels could be distinguished during observation. However, this result
is consistent with our earlier failure to observe spt-expressing neurons more rostral than the 5th most rostral somite pair (see Figure 4 and above).

At first glance, the numbers of spt-expressing neurons we observe at each somite level (i.e. per two hemisegments) at
24 hpf does not appear to be comparable to the previous observations of Bernhardt
et al. in 1990 [12] of 0.06 DoLA interneurons per hemisegment (0.12 per somite level) flanked by the
5th- to 8th-formed somite pairs in embryos at 18 hpf. However, the fact that we rarely observe
spt-expressing neurons anterior to the 6th-formed somite pair at 24 hpf combined with the possibility that these neurons migrate
rostrally after birth (see above) suggests that fewer DoLA neurons may be found in
the region flanked by the 5th- to 8th-formed somite pairs at 18 hpf compared to 24 hpf. Also, these authors identified
DoLA neurons by their pattern of arborisation whereas we have identified these cells
by spt expression. At 18 hpf many spt-expressing cells may not yet have developed characteristic DoLA arborisation patterns.
In contrast, in a study of GABAergic DoLA neurons in embryos at 27 hpf by Bernhardt
et al. in 1992 [24], a mean of 3.89 cells (standard deviation 1.17) were observed in the region of hemisegments
6 to 10. At 24 hpf, we observed a mean of 3.64 cells (standard deviation 1.08) in
the same region. The close correspondence of these figures supports that spt-expressing neural cells are DoLA neurons.

Discussion

The identity of spt-expressing neural cells

The spt-expressing cells in the developing spinal cord show coexpression of a number of neural
markers such as huC and the islet genes. This, together with the position of these cells just ventral to the Rohon-Beard
neurons and their rostrocaudal distribution establishes that these cells are likely
to be the DoLA neurons originally described by Bernhardt et al. [12]. Indeed, we are able to observe a rostrally projecting process of these cells similar
to the ascending axon of DoLA neurons due to the active transport of spt mRNA along this process.

That DoLA neurons express spt conflicts with observations of the expression in Xenopus embryos of the spt orthologous gene, Xombi. Xombi is transcribed in a very similar pattern to spt in the developing spinal cord. In 1996, Stennard et al. [25] and Zhang and King [26] suggested that this gene (they named it Antipodean and VegT respectively) might be expressed in Rohon-Beard neurons based on the dorsal/dorsolateral
position of expressing cells in the spinal cord. However, in a simultaneous publication,
Lustig et al. [27] suggested that Xombi expression was in the dorsolateral area of interneuron formation. We expect that
closer examination will show that Xombi is expressed in Xenopus DoLA-equivalent cells, probably dorsolateral interneurons (see review by Roberts
[28]).

The observation of spt mRNA in an anterior growth process/axon suggests a number of possibilities. First,
the mRNA may not be translated but may perform some other (or no) role in the process.
Second, Spt protein may be required in this process for a function other than gene
regulation. Third, spt mRNA may be required in the process for production of protein that is used to signal
back to the nucleus. There is some precedence for the expression of transcription
factors in neurites since these are known to be found in dendrites where it is thought
that they may be involved in activities such as long term potentiation [29]. Finally, and most intriguing, is the possibility that spt mRNA might be involved in signalling to cells with which the process makes contact.
It has been demonstrated that the transcription factor Engrailed and the homeodomains
of other proteins can be transported between cells [30-33]. Testing of these possibilities will require observation of the distribution of Spt
protein.

spt-expressing DoLA neurons possibly migrate rostrally

Higher numbers of spt-expressing neurons are observed rostrally compared to caudally in the spinal cord.
It may be that spt-expressing neurons continue to be born as the developing CNS matures in a rostral
to caudal progression or that caudal neurons undergo programmed cell death. However,
the marginal change in the number of these neurons between 24 and 30 hpf argues against
this. Also, ipsilateral juxtaposition of these neurons is more common at rostral compared
to caudal sites. The increased juxtaposition rostrally could be caused by rostral
migration of spt-expressing neurons when an anterior limit exists for the migration. spt-expressing neurons are rarely seen anterior of the 6th-formed somite pair suggesting that this position on the rostrocaudal axis may represent
such a limit.

spt is expressed in somitic mesoderm

Extended staining for spt mRNA revealed that this gene is transcribed at low levels in the dorsocaudal cells
of recently formed somites. It has previously been assumed that spt expression marks only presomitic mesoderm. The function of spt expression in these somitic cells is unknown. Discovery of other genes expressed
in a similar pattern in newly formed somites may reveal more of the function or fate
of these cells.

The irregular pattern of spt-expressing neurons may be based on an underlying regularity

The question of how irregular patterns of cell distribution or gene expression are
controlled is not commonly addressed in studies of developmental biology. Nevertheless,
these patterns are common in the central nervous systems of most animals and occur
in many other tissues. In the spinal cord of the developing embryo, Rohon-Beard neurons
occur at a frequency of more than one per hemisegment [12]. Their positions are not highly ordered and do not depend upon signals from mesoderm
[5]. Instead, short-range intercellular interactions controlled by Notch signalling appear
to play a role in their differentiation from a field of progenitor cells [10,11].

The ascending commissural neurons that are located just ventral to Rohon-Beard neurons
are also found at a frequency of more than one per hemisegment. However, subclasses
of these neurons exist with lower frequency. For example, anti-CON1 antibody labels
a subclass of ascending commissural neurons in the embryo that probably become commissural
primary ascending (CoPA) interneurons in the larva. These are present in an irregular
pattern on the rostrocaudal axis at a frequency of 0.87 per hemisegment flanking the
6th- to 11th-formed somite pairs at 28 hpf [12]. Ascending commissural neurons are located at a similar dorsoventral level to the
DoLA neurons. We have shown that val expression in the spinal cord occurs just ventral to spt-expressing neurons. Thus, it is possible that val labels a subclass of ascending commissural neurons.

The neuromasts of the posterior lateral line – while part of the peripheral nervous
system – are, nevertheless, an example of a neural cell type distributed at a frequency
of less than one per hemisegment. These neurons are deposited by the migrating lateral
line primordia along the myoseptum at the boundary between somites at four or five
positions along each side of the embryo. While their rostrocaudal distribution is
not completely irregular, there is considerable variation in the actual position of
any one neuromast. The position at which a neuromast is deposited appears to depend
more strongly on the distance from the previously deposited neuromast rather than
the precise position on the rostrocaudal axis [34]. Interestingly, the recessive, homozygous viable mutation hypersensitive (hps) results in neuromast deposition at nearly every somite boundary [35]. The fact that this (presumably) loss-of-function mutation can increase the regularity
of a pattern indicates that the distribution of neuromasts probably results from the
combined effect of at least two patterning mechanisms – one controlling inter-neuromast
distance and one controlling neuromast localisation to intersomitic boundaries. This
raises the question as to whether mutations might exist that increase the frequency
of generation of spt-expressing neurons, for example, by increasing the strength of a patterning signal
from the mesoderm to the developing CNS.

The dorsoventral and rostrocaudal correspondence of spt expression in newly formed somites and the CNS suggests a functional connection between
the spt expression in these two tissues. The somitic and neural cells may be responding to
a common patterning signal. Alternatively, the somitic spt expression may mark the source of a signal from the somite to neural tissue. A precedent
for the latter alternative exists in the influence of flanking mesoderm on primary
motoneuron formation [5-7]. However, the formation of most primary motoneurons occurs with complete regularity
(one neuron per hemisegment). An interesting exception to this is the Variable Primary
(VaP) motoneuron that occurs at a frequency of less than 0.5 per hemisegment. VaPs
arise adjacent to Caudal Primary (CaP) motoneurons midway between hemisegment boundaries
[36]. VaPs normally extend an axon to the horizontal myoseptum in the myotomes after which
the VaP dies. In contrast, the CaP axon continues from the myoseptum into ventral
muscle. These two neurons actually represent an equivalence pair since ablation of
a CaP causes the neighbouring VaP to develop a CaP-like arborisation pattern [37]. Thus, rather than VaP formation occurring with less than complete regularity, we
can regard this situation as CaP formation at greater than one cell per hemisegment
followed by regulation to one cell per hemisegment.

We suggest that the spt-expressing DoLA interneurons might be "inefficiently" patterned by flanking somitic
mesoderm. Thus, the initial distribution of these neurons would represent an incomplete
pattern based on a regular template. Migration and tissue growth might then scramble
this pattern. We are currently testing this hypothesis by examining the role of spt expression and mesodermal signals in DoLA neuron differentiation and distribution.

Conclusions

spt-expressing cells in the developing central nervous system appear to be DoLA interneurons.
The irregular distribution of these cells along the rostrocaudal axis of the spinal
cord may be due to "inefficient" patterning of neural spt expression by flanking, regularly distributed somites also expressing spt. Rostral migration of spt-expressing neurons might then scramble any residual regularity in their distribution.
The idea that irregular patterns of neuron distribution may arise in partial correspondence
to regular templates is a parsimonious explanation for the evolution of such patterns.

Materials and Methods

Double whole mount in situ transcript hybridisation

(Cloning of probe sources)

A cDNA clone, (26 M), corresponding to transcription from spt was isolated in a whole mount in situ transcript hybridisation screen of zebrafish embryos [38]. cDNAs corresponding to parts of transcripts from the genes huC, isl-2 and valentino were amplified by RT-PCR from embryos at 24 hpf using the oligonucleotide primers
described in Table 2. All cDNA fragments were cloned into the pGEMT vector (Promega Corporation, Madison,
WI, USA). The inserts of these clones were amplified by PCR using M13 primers and
then transcribed with T3 or SP6 RNA polymerase to produce digoxigenin- or fluorescein-labelled
antisense RNA probes (see [38]). The clones for production of probes against isl-1 and isl-3 transcripts were obtained from Hitoshi Okamoto [6,7].

Double whole mount in situ transcript hybridisation was performed essentially as described in [39] but the first staining reaction was with BCIP/NBT, inactivation of the first alkaline
phosphatase staining reaction was by heating to 65°C for 45 min in PBS and the second
staining reaction used the Alkaline Phosphatase Substrate Kit I (Vector Laboratories
Inc., Burlingame, CA, USA).

Staining for the presence of spt transcript and acetylated tubulin was performed essentially as described above for
double whole mount in situ transcript hybridisation except that spt staining using the Alkaline Phosphatase Substrate Kit I ("Vector Red", Vector Laboratories
Inc.) was performed first followed by washing for 10 min in 100 mM Tris HCl pH 8.5
then 10 min in PBS + 0.1% Tween 20 (Sigma, St. Louis, MO, USA) (PBT) before fixation
in 4% formaldehyde in PBT. Embryos were then washed 4 × 5 min in PBT, then 3 h in
PBT + 0.3% IPEGAL (Sigma) (PBTI) + 2% BSA (Fraction V, Sigma), then 1 h in PBTI +
2% BSA at 4°C before incubation overnight at 4°C in a 1:2500 dilution of anti-Acetylated
Tubulin antibody (Sigma Cat. No. T6793) in PBTI + 0.2% BSA. Embryos were then washed
6 × 1 h in PBTI then 2 × 30 min in PBTI + 2% BSA before incubation overnight at 4°C
in a 1:200 dilution of anti-mouse IgG labelled with Alexa Fluor 488 (Molecular Probes
Inc., Eugene, OR, USA) in PBTI + 0.2% BSA. Finally, embryos were washed 7 × 1 h in
PBTI before equilibration with 80% glycerol in PBT before imaging. Note that all wash
series were preceded by three rinses in the wash solution and were at room temperature
unless otherwise indicated.

Observation and statistical analysis of cell distribution

Embryos were dechorionated at 15–18 hpf, 22 hpf, 24 hpf or 30 hpf and fixed in 4%
formaldehyde in PBS at 4°C before in situ transcript hybridisation with a probe for spt. To ensure observation of all cells expressing spt including any expressing spt at low levels, the staining reaction was allowed to proceed overnight at 4°C before
the embryos were fixed in 4% formaldehyde in PBS and then equilibrated with 80% glycerol.

Light field observation of the embryos was conducted under a Zeiss Axiophot™ microscope
(Carl Zeiss Jena GmbH, Jena, Germany) at 200× magnification using DIC optics. For
examination of cell positions, the trunk-tail region of an embryo was removed from
the rest of the body and then laid flat on a slide. Photographs were taken such that
the intersomitic boundaries and the spt-expressing neural cells were simultaneously visible. Confocal imaging of embryos
was conducted on a Bio-Rad MRC-1000 UV Confocal Laser Scanning Microscope System (Bio-Rad
Laboratories Inc., Hercules, CA, USA) using a Nikon Diaphot 300 inverted microscope
(Nikon Instech Co., Ltd., Kawasaki, Kanagawa, Japan). Fluorescence was observed using
a krypton/argon laser with excitation at 488/10 nm and emission at 522/35 nm excitation
for Alexa 488 and with excitation at 568/10 nm and emission at 605/32 nm for Vector
Red. Images were processed with Adobe Photoshop version 5.0 (Adobe Systems Inc. San
Jose, California, USA) and Confocal Assistant version 4.02 (Todd Clark Brelje).

List of abbreviations used

BCIP, 5-Bromo-4-chloro-3-indolyl-phosphate.p-toluidine-salt

BSA, bovine serum albumin

CaP, Caudal Primary

CNS, central nervous system

CoPA, Commissural Primary Ascending

DIC, differential interference contrast

DLF, dorsal longitudinal fasciculus

DoLA, dorsal longitudinal ascending

isl, islet

hpf, hours post fertilisation

NBT, Nitroblue tetrazolium chloride

PBS, phosphate buffered saline

PBT, PBS + 0.1% Tween 20

PBTI, PBT + 0.3% IPEGAL

RT-PCR, reverse transcription polymerase chain reaction

spt, spadetail

val, valentino

VaP, Variable Primary

Authors' contributions

RT carried out the majority of the in situ transcript hybridisation, cell counting, and statistical analyses and some photography.

SW performed the in situ transcript hybridisation analyses on 12 – 16 hpf embryos and staining for acetylated
tubulin.

JGC contributed to the statistical analysis

ML directed the research, performed observation of staining patterns, some cell counting
and statistical analysis, some photography and drafted the manuscript.

Acknowledgements

The authors wish to thank Dan Kortschak and Judith Eisen for valuable discussion and
Simon Koblar for critical reading of the manuscript. However, the conclusions drawn
by the authors are theirs alone. We thank Meredith Wallwork for assistance with confocal
microscopy. Clones for production of probes against the isl-1 and isl-3 genes were the kind gift of Hitoshi Okamoto. This work was supported by an Australian
Research Council small grant and by funds from the Special Research Centre for the
Molecular Genetics of Development. RT was supported by an International Postgraduate
Research Scholarship from The University of Adelaide.